Thursday, December 27, 2012

Gene Mapping by In Situ Hybridization

Gene Mapping by In Situ Hybridization

The previous mapping methods are indirect in that they provide information on the physical location of a gene on a particular chromosome but without actually visualizing the gene's map position. A more direct approach is in situ hybridization, which involves hybridizing DNA (or RNA) probes directly to metaphase chromosomes spread on a slide and visualizing the hybridization signal (and thus the location of the gene to which the probe hybridizes) under a microscope.

The DNA in metaphase chromosomes is denatured in place (hence, in situ) on the slide, and hybridization of a labeled probe is allowed to proceed. Methods for mapping single-copy gene sequences by in situ hybridization originally were laborious and slow, requiring long exposures of the slides under photographic emulsion to detect the location of hybridized probe that had been labeled with low-level isotopes, such as tritium. Mapping with confidence required analysis of many metaphase spreads to distinguish the real hybridization signal from background radioactivity. However, more sensitive techniques have now been developed that enable rapid detection of hybridized probes labeled non radioactively with compounds that can be visualized by fluorescence microscopy (Fig). Even in a single metaphase spread, one can easily see the position of the gene being mapped.

In combination with banding methods for chromosome identification, fluorescence in situ hybridization can be used to map genes to within 1 to 2 million base pairs (1000 to 2000 kb) along a metaphase chromosome. Although this degree of resolution is a considerable improvement over other methods, it is still substantially larger than the size of most individual genes.




Figure: Gene mapping by in situ hybridization of a biotin-labeled DNA probe for the human muscle glycogen phosphorylase gene (MGP) to a spread of human metaphase chromosomes. Location of the MGP gene is indicated by the bright spots seen over each chromatid at the site of the gene in band q13 of chromosome 11. The mapping of MGP to 11q13 also assigns the locus for McArdle disease, an autosomal recessive myoglobinuria caused by deficiency of MGP. (Photograph courtesy of Peter Lichter, Yale University)

Celera Genomics HGP

Celera Genomics & HGP

In 1998, an identical, privately funded quest was launched by the American researcher Craig Venter and his firm Celera Genomics. The $300 million Celera effort was intended to proceed at a faster pace and at a fraction of the cost of the roughly $3 billion publicly-funded project.Celera Genomics was established in May 1998 by the Perkin-Elmer Corporation (and was later purchased by Applera Corporation), with Dr. J. Craig Venter from The Institute for Genomic Research (TIGR) as its first president. While at TIGR, Venter and Hamilton Smith led the first successful effort to sequence an entire organism's genome, that of the Haemophilus influenzae bacterium. Celera was formed for the purpose of generating and commercializing genomic information to accelerate the understanding of biological processes.
The rise and fall of Celera as an ambitious competitor of the Human Genome Project is the main subject of the book The Genome War by James Shreeve, who takes a strong pro-Venter point of view. (He followed Venter around for two years in the process of writing the book.) A view from the public effort's side is that of Nobel laureate Sir John Sulston in his book The Common Thread: A Story of Science, Politics, Ethics and the Human Genome.Celera used a newer, riskier technique called whole genome shotgun sequencing, which had been used to sequence bacterial genomes up to 6 million base pairs in length, but not for anything nearly as large as the 3 billion base pair human genome.Celera initially announced that it would seek patent protection on "only 200-300" genes, but later amended this to seeking "intellectual property protection" on "fully-characterized important structures" amounting to 100-300 targets. Contrary to its public promises, the firm eventually filed patent applications on 6,500 whole or partial genes.Although the working draft was announced in June 2000, it was not until February 2001 that Celera and the HGP scientists published details of their drafts. Special issues of Nature (which published the publicly-funded project's scientific paper) and Science (which published Celera's paper) described the methods used to produce the draft sequence and offered analysis of the sequence. These drafts covered about 90% of the genome, with much of the remaining 10% filled in later. In February 2001, at the time of the joint publications, press releases announced that the project had been completed by both groups. Improved drafts were announced in 2003 and again in 2005, filling in roughly 8% of the remaining sequence.
HGP is the most well known of many international genome projects aimed at sequencing the DNA of a specific organism. While the human DNA sequence offers the most tangible benefits, important developments in biology and medicine are predicted as a result of the sequencing of model organisms, including mice, fruit flies, zebrafish, yeast, nematodes, plants, and many microbial organisms and parasites.In 2005, researchers from the International Human Genome Sequencing Consortium (IHGSC) of the HGP announced a new estimate of 20,000 to 25,000 genes in the human genome. Previously 30,000 to 40,000 had been predicted, while estimates at the start of the project reached up to as high as 2,000,000. The number continues to fluctuate and it is now expected that it will take many years to agree on a precise value for the number of genes in the human genome.

RNA genes


RNA genes

RNA genes (sometimes referred to as non-coding RNA or small RNA) are genes that encode RNA that is not translated into a protein. The most prominent examples of RNA genes are transfer RNA (tRNA) and ribosomal RNA (rRNA), both of which are involved in the process of translation. However, since the late 1990s, many new RNA genes have been found, and thus RNA genes may play a much more significant role than previously thought. In the late 1990s and early 2000, there has been persistent evidence of more complex transcription occurring in mammalian cells (and possibly others). This could point towards a more widespread use of RNA in biology, particularly in gene regulation. A particular class of non-coding RNA, micro RNA, has been found in many metazoans (from Caenorhabditis elegans to Homo sapiens) and clearly plays an important role in regulating other genes. First proposed in 2004 by Rassoulzadegan and published in Nature 2006.

RNA is implicated as being part of the germline. If confirmed, this result would significantly alter the present understanding of genetics and lead to many question on DNA-RNA roles and interactions.RNA Deatiles,ScienceRibonucleic acid (RNA) is a nucleic acid polymer consisting of nucleotide monomers, that acts as a messenger between DNA and ribosomes, and that is also responsible for making proteins out of amino acids. RNA polynucleotides contain ribose sugars and predominantly uracil unlike deoxyribonucleic acid (DNA), which contains deoxyribose and predominantly thymine. It is transcribed (synthesized) from DNA by enzymes called RNA polymerases and further processed by other enzymes.

RNA serves as the template for translation of genes into proteins, transferring amino acids to the ribosome to form proteins, and also translating the transcript into proteins. Nucleic acids were discovered in 1868 (some sources indicate 1869) by Johann Friedrich Miescher (1844-1895), who called the material 'nuclein' since it was found in the nucleus. It was later discovered that prokaryotic cells, which do not have a nucleus, also contain nucleic acids. The role of RNA in protein synthesis had been suspected since 1939, based on experiments carried out by Torbjörn Caspersson, Jean Brachet and Jack Schultz. Hubert Chantrenne elucidated the messenger role played by RNA in the synthesis of proteins in ribosome.

The sequence of the 77 nucleotides of a yeast RNA was found by Robert W. Holley in 1964, winning Holley the 1968 Nobel Prize for Medicine. In 1976, Walter Fiers and his team at the University of Ghent determined the complete nucleotide sequenceDNA Bases Bio TechnologyDeoxyribonucleic acid, or DNA is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all living organisms. The main role of DNA is the long-term storage of information and it is often compared to a set of blueprints, since DNA contains the instructions needed to construct other components of cells, such as proteins and RNA molecules.
The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information. Chemically, DNA is a long polymer of simple units called nucleotides, which are held together by a backbone made of alternating sugars and phosphate groups. Attached to each sugar is one of four types of molecules called bases. It is the sequence of these four bases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA, in a process called transcription. Most of these RNA molecules are used to synthesize proteins, but others are used directly in structures such as ribosomes and spliceosomes.
Within cells, DNA is organized into structures called chromosomes and the set of chromosomes within a cell make up a genome. These chromosomes are duplicated before cells divide, in a process called DNA replication. Eukaryotic organisms such as animals, plants, and fungi store their DNA inside the cell nucleus, while in prokaryotes such as bacteria it is found in the cell's cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA, which helps control its interactions with other proteins and thereby control which genes are transcribed Biotechnology Indroduction. The convention recognized for the first time in international law that the conservation of biological diversity is "a common concern of humankind" and is an integral part of the development process.

The agreement covers all ecosystems, species, and genetic resources. It links traditional conservation efforts to the economic goal of using biological resources sustainably. It sets principles for the fair and equitable sharing of the benefits arising from the use of genetic resources, notably those destined for commercial use. It also covers the rapidly expanding field of biotechnology through its Cartagena Protocol on Biosafety, addressing technology development and transfer, benefit-sharing and biosafety issues. Importantly, the Convention is legally binding; countries that join it('Parties') are obliged to implement its provisions .

Apply Bio Technology Science The convention reminds decision-makers that natural resources are not infinite and sets out a philosophy of sustainable use. While past conservation efforts were aimed at protecting particular species and habitats, the Convention recognizes that ecosystems, species and genes must be used for the benefit of humans. However, this should be done in a way and at a rate that does not lead to the long-term decline of biological diversity The convention also offers decision-makers guidance based on the precautionary principle that where there is a threat of significant reduction or loss of biological diversity, lack of full scientific certainty should not be used as a reason for postponing measures to avoid or minimize such a threat. The Convention acknowledges that substantial investments are required to conserve biological diversity. It argues, however, that conservation will bring us significant environmental, economic and social benefits in return.In this situation, your range of choices is very broad and many packages will meet these limited.

Fermentation

Fermentation

The word fermentation is derived from a latin verb ‘fervere’ which means to boil. However, events of boiling came into existence from the fact that during alcoholic fermentation, the bubbles of gas (CO2) burst at the surface of a boiling liquid and give the warty appearance. The conventional definition of fermentation is the breakdown (metabolism) of larger molecules. For example, carbohydrates, into simple ones under the influence of micro-organism for their enzymes. This definition of fermentation had little meaning until the metabolic process were known. In a micro-biological way, fermentation is defined as “any process for the production of useful products through mass culture of micro-organism” wheteras, in a biochemical sense, this word means the numerous oxidation – reduction reactions in which organic compounds, used as source of carbon and energy, act as acceptors of donors of hydrogen ions. The organic compounds used as substrate give rise to various products of fermentation which accumulate in the growth medium (Riviere, 1977)

Almost in all organism metabolic pathways generating energy are fundamentally similar. In autophototrophs, (e.g. some bacteria, cyanobacteria and higher plants) ATP is generated as a result of photosynthesis electron transport mechanisms, whereas in chemotrophs the source of ATP is oxidation of organic compounds in the growth substrates. The oxidation reaction may be accomplished in the presence of oxygen (in aerobes) or in absence of oxygen (in anaerobes). Thus, in aerobic microorganism the process in ATP generation is referred to as cellular respiration whereas in anaerobes or aerobes functioning under anaerobic condition, it is known as anaerobic respiration or fermentation.

Although, fermentation (e.g. brewing and wine production) was done for many hundred years, yet during the end of 15th century, brewing became partially industrialized in Britain. Antony van Leeuwenhoek (1632-1723) developed method to observe yeasts and other micro-organism under the microscope but this study could not be further strengthened. By early 19th century Cagniard-Latour and Schwann reported that the fermentation of wine and beer is accomplished by yeast cells. It was L. Pasteur who observed microorganism associated with fermentation and causing many diseases in human beings. Detailed studies on fermentation products, culture improvement, recovery, and scale up of products were made after the world war I.

The Human Genome Projects - Benefits

The Human Genome Projects - Benefits

The work on interpretation of genome data is still in its initial stages. It is anticipated that detailed knowledge of the human genome will provide new avenues for advances in medicine and biotechnology. Clear practical results of the project emerged even before the work was finished. For example, a number of companies, such as Myriad Genetics started offering easy ways to administer genetic tests that can show predisposition to a variety of illnesses, including breast cancer, disorders of hemostasis, cystic fibrosis, liver diseases and many others. Also, the etiologies for cancers, Alzheimer's disease and other areas of clinical interest are considered likely to benefit from genome information and possibly may lead in the long term to significant advances in their management.
There are also many tangible benefits for biological scientists. For example, a researcher investigating a certain form of cancer may have narrowed down his search to a particular gene. By visiting the human genome database on the worldwide web, this researcher can examine what other scientists have written about this gene, including (potentially) the three-dimensional structure of its product, its function(s), its evolutionary relationships to other human genes, or to genes in mice or yeast or fruit flies, possible detrimental mutations, interactions with other genes, body tissues in which this gene is activated, diseases associated with this gene or other datatypes.Further, deeper understanding of the disease processes at the level of molecular biology may determine new therapeutic procedures. Given the established importance of DNA in molecular biology and its central role in determining the fundamental operation of cellular processes, it is likely that expanded knowledge in this area will facilitate medical advances in numerous areas of clinical interest that may not have been possible without them.The analysis of similarities between DNA sequences from different organisms is also opening new avenues in the study of the theory of evolution. In many cases, evolutionary questions can now be framed in terms of molecular biology; indeed, many major evolutionary milestones (the emergence of the ribosome and organelles, the development of embryos with body plans, the vertebrate immune system) can be related to the molecular level. Many questions about the similarities and differences between humans and our closest relatives (the primates, and indeed the other mammals) are expected to be illuminated by the data from this project.
The Human Genome Diversity Project, spin-off research aimed at mapping the DNA that varies between human ethnic groups, which was rumored to have been halted, actually did continue and to date has yielded new conclusions. In the future, HGDP could possibly expose new data in disease surveillance, human development and anthropology. HGDP could unlock secrets behind and create new strategies for managing the vulnerability of ethnic groups to certain diseases (see race in biomedicine). It could also show how human populations have adapted to these vulnerabilities.

What's Turning Genomics Vision Into Reality

In "A Vision for the Future of Genomics Research," published in the April 24, 2003 issue of the journal Nature, the National Human Genome Research Institute (NHGRI) details a myriad of research opportunities in the genome era. This backgrounder describes a few of the more visible, large-scale opportunities.

The International HapMap Project

Launched in October 2002 by NHGRI and its partners, the International HapMap Project has enlisted a worldwide consortium of scientists with the goal of producing the "next-generation" map of the human genome to speed the discovery of genes related to common illnesses such as asthma, cancer, diabetes and heart disease.Expected to take three years to complete, the "HapMap" will chart genetic variation within the human genome at an unprecedented level of precision. By comparing genetic differences among individuals and identifying those specifically associated with a condition, consortium members believe they can create a tool to help researchers detect the genetic contributions to many diseases. Whereas the Human Genome Project provided the foundation on which researchers are making dramatic genetic discoveries, the HapMap will begin building the framework to make the results of genomic research applicable to individuals.

ENCyclopedia Of DNA Elements (ENCODE)

This NHGRI-led project is designed to develop efficient ways of identifying and precisely locating all of the protein-coding genes, non-protein-coding genes and other sequence-based, functional elements contained in the human DNA sequence. Creating this monumental reference work will help scientists mine and fully utilize the human sequence, gain a deeper understanding of human biology, predict potential disease risk, and develop new strategies for the prevention and treatment of disease.The ENCODE project will begin as a pilot, in which participating research teams will work cooperatively to develop efficient, high-throughput methods for rigorously and fully analyzing a defined set of target regions comprising approximately 1 percent of the human genome. Analysis of this first 30 megabases (Mb) of human genome sequence will allow the project participants to test and compare a variety of existing and new technologies to find the functional elements in human DNA.

Chemical Genomics

NHGRI is exploring the acquisition and/or creation of publicly available libraries of organic chemical compounds, also referred to as small molecules, for use by basic scientists in their efforts to chart biological pathways. Such compounds have a number of attractive features for genome analysis, including their wide structural diversity, which mirrors the diversity of the genome; their ability in many cases to enter cells readily; and the fact that they can often serve as starting points for drug development. The use of these chemical compounds to probe gene function will complement more conventional nucleic acid approaches.This initiative offers enormous potential. However, it is a fundamentally new approach to genomics, and largely new to basic biomedical research as a whole. As a result, substantial investments in physical and human capital will be needed. NHGRI is currently planning for these needs, which will include large libraries of chemical compounds (500,000 - 1,000,000 total); capacity for robotic-enabled, high-throughput screening; and medicinal chemistry to convert compounds identified through such screening into useful biological tools.

Genomes to Life

The Department of Energy's "Genomes to Life" program focuses on single-cell organisms, or microbes. The fundamental goal is to understand the intricate details of the life processes of microbes so well that computational models can be developed to accurately describe and predict their responses to changes in their environment."Genomes to Life" aims to understand the activities of single-cell organisms on three levels: the proteins and multi-molecular machines that perform most of the cell's work; the gene regulatory networks that control these processes; and microbial associations or communities in which groups of different microbes carry out fundamental functions in nature. Once researchers understand how life functions at the microbial level, they hope to use the capabilities of these organisms to help meet many of our national challenges in energy and the environment.

Structural Genomics Consortium

Structural genomics is the systematic, high-throughput generation of the three-dimensional structure of proteins. The ultimate goal for studying the structural genomics of any organism is the complete structural description of all proteins encoded by the genome of that organism. Such three-dimensional structures will be crucial for rational drug design, for diagnosis and treatment of disease, and for advancing our understanding of basic biology. A broad collection of structures will provide valuable biological information beyond that which can be obtained from individual structures.